Anti-apoptotic Bcl-2 Family Members Research Articles

Abstract B30: Mechanisms of resistance to ABT-199 in leukemia and lymphoma cell lines

Abstract ABT-199 is a first-in-class orally bioavailable BCL-2 selective inhibitor that is currently being testing in clinical trials. Clinical activity has been reported in chronic lymphocytic leukemia and NHL patients treated with ABT-199 as a monotherapy. As with any targeted cancer therapy, it is important to identify the potential mechanisms of resistance, not only to inform patient selection but also to develop strategies to circumvent resistance as it emerges. In this study, we generated resistant variants from ABT-199-sensitive cell lines of different leukemia and lymphoma subtypes (two diffuse large B-cell lymphomas, two follicular lymphomas, one leukemia line, and two mantle cell lymphomas) by increasing exposure to ABT-199 in a stepwise manner over time. Compared to the parental cell lines, the variants were 10 to >100-fold less sensitive to ABT-199. The ABT-199-resistant variants were also resistant to ABT-263 (navitoclax), a BCL-2/BCL-XL inhibitor, as well as to other standard-of-care cancer agents. Gene and protein expression analyses revealed multiple alterations in the expression of BCL-2 family members in the resistant variants; however, not all of the changes were universal. Alterations included changes in the levels of the target protein BCL-2, reduction of the pro-apoptotic proteins BAX, BIM or NOXA, or increases in the anti-apoptotic proteins BCL-XL or MCL-1. In some but not all cases, the changes to gene and protein expression levels were accompanied by changes in DNA copy number. To determine whether changes in the anti-apoptotic BCL-2 family members were causally related to ABT-199 resistance, we tested ABT-199 in combination with BCL-XL and MCL-1 selective inhibitors. Co-treating with ABT-199 and either a BCL-XL or MCL-1 selective compound was synergistic in killing the ABT-199-resistant variants, suggesting these proteins can both contribute directly to ABT-199 resistance. Under appropriate selection pressure, intrinsic traits that mitigate the effects of anticancer drugs can lead to the enrichment of a resistant tumor population. In ABT-199-resistant RS4;11 cells we observed a complete and permanent loss of BAX, one of the ultimate effectors of mitochondrial mediated apoptosis. FACS analysis revealed that there was a pre-existing population of RS4;11 cells lacking this protein. It is likely that these cells had a selective advantage over those expressing BAX when treated with ABT-199. Overall our data indicate that there are distinct mechanisms of resistance to ABT-199, either innate or acquired, which will have important implications for both patient selection and developing strategies to overcome resistance. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for the research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Stephen K. Tahir, Morey L. Smith, Paul Hessler, Lisa Roberts-Rapp, Joel D. Leverson, Lloyd T. Lam. Mechanisms of resistance to ABT-199 in leukemia and lymphoma cell lines. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B30.

Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance?

Apoptosis, cell death executed by caspases, is essential to normal breast development and homeostasis. Pro-apoptotic and anti-apoptotic signals are tightly regulated in normal breast epithelial cells. Dysregulation of this balance is required for breast tumorigenesis and increases acquired resistance to treatments, including molecularly targeted therapies, radiation and chemotherapies. The pro-apoptotic or anti-apoptotic Bcl-2 family members interact with each other to maintain mitochondrial integrity and regulate cellular commitment to apoptosis. Among the anti-apoptotic Bcl-2 family members, Mcl-1 is uniquely regulated by numerous oncogenic signaling pathways. This review will focus on the role of Bcl-2 family proteins in normal breast development, breast tumorigenesis and acquired resistance to breast cancer treatment strategies, while highlighting Mcl-1 as a promising target to improve breast cancer tumor cell killing.

Knockdown of the antiapoptotic Bcl-2 family member A1/Bfl-1 protects mice from anaphylaxis.

Many forms of hypersensitivity reactions and allergic responses depend on deregulated mast cell activity. Several reports suggested that the antiapoptotic Bcl-2 family protein Bcl2a1/Bfl-1/A1 plays a critical role in mast cell survival upon activation. However, its in vivo relevance is poorly understood because of quadruplication of the Bcl2a1 gene locus in mice, hindering conventional knockout studies. In this study, we used a mouse model allowing traceable constitutive knockdown of all A1 isoforms expressed in the hematopoietic system by RNA interference. Knockdown of A1 reduced mast cell numbers in the skin and impaired connective tissue-like mast cell survival upon FcεRI-mediated activation in vitro. In contrast, A1 was dispensable for mucosa-like mast cell differentiation and survival. Moreover, knockdown of A1 prevented IgE-mediated passive systemic and cutaneous anaphylaxis in vivo. Our findings demonstrate that A1 is essential for the homeostasis of connective tissue mast cells, identifying A1 as a possible therapeutic target for therapy of certain types of mast cell-driven allergy symptoms.

Antihelminthic benzimidazoles potentiate navitoclax (ABT-263) activity by inducing Noxa-dependent apoptosis in non-small cell lung cancer (NSCLC) cell lines.

BackgroundEvasion of apoptosis is a hallmark of cancer cells. One mechanism to deregulate the apoptotic pathway is by upregulation of the anti-apoptotic Bcl-2 family members. Navitoclax (ABT-263) is a Bcl-2/Bcl-xL inhibitor that restores the ability of cancer cells to undergo apoptosis.MethodsIn this study we performed a high-throughput screen with 640 FDA-approved drugs to identify potential therapeutic combinations with navitoclax in a non-small cell lung cancer (NSCLC) cell line.ResultsOther than a panel of cancer compounds such as doxorubicin, camptothecin, and docetaxel, four antihelminthic compounds (benzimidazoles) potentiated navitoclax activity. Treatment with benzimidazoles led to induction of the pro-apoptotic protein Noxa at the mRNA and protein level. Noxa binds and antagonizes antiapoptotic protein Mcl-1. siRNA-mediated knock-down of Noxa completely rescued benzimidazole-potentiated navitoclax activity. In addition, inhibiting caspase 3 and 9 partially rescued benzimidazole-potentiated navitoclax activity.ConclusionsWe have identified compounds and mechanisms which potentiate navitoclax activity in lung cancer cell lines. Further validation of the benzimidazole-potentiated navitoclax effect in vivo is required to evaluate the potential for translating this observation into clinical benefit.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-014-0151-3) contains supplementary material, which is available to authorized users.

Abstract B18: Blocking α4β1 integrin interaction with matrix metalloproteinase-9 overcomes stroma-induced resistance of chronic lymphocytic leukemia cells to fludarabine and arsenic trioxide

Abstract Chronic lymphocytic leukemia (CLL) progression is determined by CLL cell accumulation in lymphoid organs, where they receive survival signals that impair therapeutic treatments. MMP-9 is a microenvironment component and we have shown that induces a signalling pathway that leads to CLL cell survival. We have now studied whether MMP-9 is involved in the response to cytotoxic agents such as fludarabine and arsenic trioxide (ATO). Both agents upregulated MMP-9 (mRNA and protein) and enhanced its binding to CLL cells. Cell-associated MMP-9 was restricted to early apoptotic cells and totally dependent on apoptosis onset, suggesting a role in the response to these drugs. Indeed, anti-MMP-9 antibodies accelerated apoptosis. Moreover, MEC-1 cells (CLL-derived) stably transfected with MMP-9 were more resistant to drug-induced apoptosis than Mock-transfected cells. Western blot analyses revealed that MMP-9 prevented down regulation of anti-apoptotic Bcl-2 family members. Therefore, MMP-9 likely represents a compensatory survival response that contributes to drug resistance. Additionally, culturing CLL cells with bone marrow stromal cells (BMSC) prevented the apoptotic effect of ATO and this was nearly completely reverted with anti-MMP-9 antibodies. Blocking α4β1 integrin (MMP-9 receptor) and/or αLβ2 integrin (ICAM-1 receptor) also overcame stroma-induced survival. This was also partially achieved by culturing CLL cells with BMSC-conditioned medium, indicating that both cell adhesion and soluble factors contributed to the observed drug resistance. To determine the mechanisms involved, the effect of blocking several signalling pathways was studied. Inhibiting the PI3K/Akt or focal adhesion pathways with specific inhibitors significantly restored the sensitivity of CLL cells to ATO. Therefore, our results highlight the role of MMP-9 in CLL drug resistance and indicate that concomitant targeting of MMP-9 may improve the CLL response to treatment. Supported by grants SAF2012-31613 and RD12/0036/0061 (AGP) from the Ministry of Economy and Innovation, Spain; S2010/BMD-2314 (AGP) from the Comunidad de Madrid/European Union, and by a grant from the Fundación Puerta de Hierro, Madrid (JAGM). Citation Format: Irene Amigo-Jiménez, Elvira Bailón, Estefanía Ugarte-Berzal, José Antonio García-Marco, Angeles Garcia-Pardo. Blocking α4β1 integrin interaction with matrix metalloproteinase-9 overcomes stroma-induced resistance of chronic lymphocytic leukemia cells to fludarabine and arsenic trioxide. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B18. doi:10.1158/1538-7445.CHTME14-B18

MCL-1 and BCL-xL-dependent resistance to the BCL-2 inhibitor ABT-199 can be overcome by preventing PI3K/AKT/mTOR activation in lymphoid malignancies.

Overexpression of anti-apoptotic BCL-2 family members is a hallmark of many lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) that can be targeted with small molecule inhibitors. ABT-199 is a rationally designed BCL-2 homology (BH)-3 mimetic that specifically binds to BCL-2, but not to MCL-1 and BCL-xL. Although the thrombocytopenia that occurs with navitoclax treatment has not been a problem with ABT-199, clinical trials in CLL could benefit by lowering the ABT-199 concentration through targeting other survival pathways. In this study, we investigated the mechanisms of resistance that develops to ABT-199 therapy by generating ABT-199-resistant (ABT199-R) cell lines via chronic exposure of NHL cell lines to ABT-199. Acquired resistance resulted in substantial AKT activation and upregulation of MCL-1 and BCL-xL levels that sequestered BIM. ABT199-R cells exhibited increased MCL-1 stability and failed to activate BAX in response to ABT-199. The ABT-199 acquired and inherent resistant cells were sensitized to treatment with ABT-199 by inhibitors of the PI3K, AKT, and mTOR pathways, NVP-BEZ235 and GS-1101. NVP-BEZ235, a dual inhibitor of p-AKT and mTOR, reduced MCL-1 levels causing BIM release from MCL-1 and BCL-xL, thus leading to cell death by BAX activation. The PI3Kδ inhibitor GS-1101 (idelalisib) downregulated MCL-1 and sensitized ABT199-R cells through AKT-mediated BAX activation. A genetic approach, through siRNA-mediated down-regulation of AKT, MCL-1, and BCL-xL, significantly decreased cell survival, demonstrating the importance of these cell survival factors for ABT-199 resistance. Our findings suggest a novel mechanism that modulates the expression and activity of pro-survival proteins to confer treatment resistance that could be exploited by a rational combination therapeutic regimen that could be effective for treating lymphoid malignancies.

Evaluation of Mcl-1 Inhibitors in Preclinical Models of Multiple Myeloma

Mcl-1, an anti-apoptotic Bcl-2 family member, is a known resistance factor to many cancer therapies, and a historically difficult target to drug. Recently however, we have made significant progress optimizing the potency and characterizing the mechanism of action of a novel class of selective Mcl-1 inhibitors. Here, we characterize the activity of one of our lead compounds and confirm on-target mechanistic activity in vitro and in vivo.Using an Mcl-1 binding assay we demonstrate very potent activity (IC50 < 3nM), while maintaining selectivity over other anti-apoptotic Bcl-2 family proteins, Bcl-2 (IC50 > 33 µM) and Bcl-xL (IC50 > 33 µM). In an Mcl-1 dependent multiple myeloma cell line, MOLP-8, we demonstrate rapid Mcl-1/Bak complex disruption with 10 nM inhibitor by 15 min followed by caspase 9-dependent apoptosis by 1 hr (EC50= 50 nM), consistent with an Mcl-1 dependent mechanism of action. Importantly, by depleting Bak from cells using siRNA, we also confirm that the observed apoptosis is through a Bak-dependent mechanism. Moreover, our Mcl-1 inhibitor exhibits broad submicromolar activity across a panel of multiple myeloma cell lines.In the absence of a validated selective Mcl-1 reference compound to benchmark in vivo activity, we engineered MOLP-8 cells to express the pro-apoptotic protein, Noxa, using a Tet-on expression system and grew these as subcutaneous tumors on the flanks of SCID mice. Within 24 hours of switching the mice to a diet containing doxycycline, we observe significant upregulation of Noxa, leading to an increase in cleaved caspase 3 (CC3) and cleaved PARP in tumor lysates, resulting in rapid tumor regression. In the same MOLP-8 xenograft model, we demonstrate that our Mcl-1 inhibitor induces rapid dissociation of Bak from Mcl-1 in tumors leading to accumulation of CC3 and cleaved PARP by 1 hr. Complete tumor regression was observed after a single 60 mg/kg or 100 mg/kg dose, while partial regression was observed after a single 30 mg/kg dose, and tumor growth inhibition was observed after a single 10 mg/kg dose. All doses were well tolerated with no significant body weight loss. Together, these data reinforce the potential utility of selective Mcl-1 inhibitors in hematological malignancies. DisclosuresBelmonte:Astrazeneca: Employment, Equity Ownership. Adam:Astrazenenca: Employment, Equity Ownership. Borrelli:AstraZeneca: Employment, Equity Ownership. Bhavsar:AstraZeneca: Employment, Equity Ownership. Bebernitz:AstraZeneca: Employment, Equity Ownership. Hird:AstraZeneca: Employment, Equity Ownership. Secrist:AstraZeneca: Employment, Equity Ownership.

GCS-100 Induces Apoptosis of Acute Myeloid Leukemia Cells By Disrupting Galectin-Mediated Survival Signaling

Galectins are a family of b-galactoside binding proteins with effects on cell adhesion, apoptosis, cell cycle, and mRNA processing. Galectin-3 (LGALS3) is unique among galectins by having an N terminal region of roughly 130 amino acids that allows for multimerization and binding to other proteins independent of carbohydrate binding. In addition to promoting BCL2 gene expression and mitochondrial integrity, LGALS3 (along with LGALS1) positively regulates RAS signaling and thus stabilizes survival proteins dependent on ERK phosphorylation such as MCL-1. The pro-survival functions of LGALS3 and other galectins suggest that their targeting could be therapeutic for cancers including AML. Indeed, LGALS3 expression is a predictor of poor prognosis in acute myeloid leukemia (AML), as reported by Cheng and colleagues (Blood 2013) for patients with non-M3 AML and CN-AML. The modified pectin GCS-100 (La Jolla Pharmaceutical, San Diego, CA), now in a Phase II clinical trial for chronic kidney disease, binds and blocks the function of LGALS3. We report that GCS-100 suppresses the growth of AML cell lines OCI-AML3, THP-1, and HL60 in vitro as a single agent, at doses under the 250 ug/mL (i.e., within clinically-achievable concentrations). Short-term treatment of cells (i.e., < 6 hr) potently suppressed phosphorylation of AKT and ERK and reduced expression of BCL2 and MCL-1. Because LGALS3 positively regulates anti-apoptotic BCL2 family members, the Raz group has suggested targeting galectins to enhance efficacy of BH3 mimetic drugs (Harazano et al Cancer Metastasis Review 2013). We found that GCS-100 potently synergized with ABT-737 to kill OCI-AML3 cells: while 1 uM ABT-737 or 125 ug/mL GCS-100 reduced total viable cells by ~ 30% and induced apoptosis in < 20% of cells after 48 hr as single agents, their combination at those doses and time point reduced viable cells by ~ 94% and induced apoptosis in ~ 70% of cells. Suppression of LGALS3 by lentiviral shRNA reduced BCL2 gene expression as determined by qRT-PCR and augmented killing with ABT-737. Lentiviral suppression of LGALS3 protected cells from GCS-100 at doses of 250 ug/mL but reduction of the galectin failed to protect cells from higher doses of the drug (i.e., 500 ug/mL). This result suggests other galectins are likely inhibited at higher doses of the agent. We used gene expression profiling (GEP) on Illumina HT12v4 human whole-genome arrays to assess more broadly the molecular effects of inhibiting galectins in AML cell lines OCI-AML3 and THP-1 treated with 250 ug/mL or 500 ug/ml GCS-100 for 24 hr. Data were analyzed by Gene Set Enrichment Analysis (GSEA) using gene sets from the Molecular Signatures Database (www.broadinstitute.org/gsea/msigdb/). GSEA suggested that GCS-100 promotes differentiation and inhibits genes associated with proliferation. Multiple upregulated gene sets suggest that there may be a release of a differentiation block as a result of GCS-100 treatment. Furthermore, two gene sets suggest that GCS-100 behaves similar to a GSK3 inhibitor: Known pathways regulated by GSK3 in hematopoietic stem cells are mTOR and Wnt/beta Catenin. Inhibition of Wnt/beta Catenin can release a differentiation block. Consistent with GCS-100 promoting cell differentiation, lentiviral shRNA reduced LGALS3 protein > 90% in THP-1 cells and increased CD11b expression, suggesting increased differentiation, compared to cells with control shRNA. GCS-100 was tested in an in vitro model of the bone marrow microenvironment using BM-derived mesenchymal stromal cell (MSC). MSC can protect leukemia cells from a variety of clinically relevant chemotherapy drugs including AraC. GCS-100 was effective at killing AML cells despite the presence of MSC. Both THP-1 and OCI-AML3 cells exhibited > 80% and > 60% reduction of viable cells, respectively, despite the presence of MSC when treated with 250 ug/mL GCS-100 for 72 hours. In addition, GCS-100 was found to block adhesion of OCI-AML3 cells to MSC suggesting that GCS-100 could be effective in mobilizing AML cells. In summary, our findings suggest that GCS-100 can induce apoptosis in AML cells as a single agent or in combination with the BH3 mimetic ABT-737. The agent is effective even in the presence of MSC suggesting it could be efficacious in the leukemia niche. These findings suggest GCS-100 could be effective for AML therapy. DisclosuresRolke:La Jolla Pharmaceutical Company: Employment. Tidmarsh:La Jolla Pharmaceutical Company: Employment.

Small Molecule ONC201/TIC10 Induces Caspase-Dependent Apoptosis in Acute Lymphoblastic Leukemia Cells Via Modulation of Bcl-2 and IAP Family Proteins

ONC201/TIC10 is a potent small molecule anti-tumor agent in several types of solid tumors and lymphomas. ONC201/TIC10 is on track to enter clinical trials for patients with advanced cancer in 2014, with IND issued by the FDA in March, 2014. Early trials will evaluate the safety and efficacy of ONC201/TIC10 as a monoagent in hematological malignancies. In the current study, we evaluated the anti-cancer effects of the small molecule in Acute Lymphoblastic Leukemia (ALL). Analysis of cell viability by the CellTiter-Glo method revealed that ONC201/TIC10 treatment reduces the viability of three ALL cell lines (Reh, Jurkat, MOLT-4) in a dose- (2.5/5/10 μM) and time-dependent manner (24/48/72 h). We have previously reported that ONC201/TIC10-mediated reduction in cell viability and apoptosis in various types of solid tumors occurs at 60/72 h. Interestingly, ONC201/TIC10 reduces the viability of ALL cell lines within 24/48 h at the indicated doses. An inactive TIC10 isomer compound synthesized by Medkoo Biosciences with a structure related to the active ONC201/TIC10 compound does not reduce the viability of ALL cells. Sub-G1 analysis indicated that ONC201/TIC10 induces apoptosis in ALL cells and a pan-caspase inhibitor reduces ONC201/TIC10-mediated apoptosis. Western blot analysis was used to further investigate the mechanism of ONC201/TIC10-mediated apoptosis. ONC201/TIC10-mediated apoptosis involves PARP cleavage and caspase-9 activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl are downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201/TIC10 treatment. ONC201/TIC10 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2. We have previously shown that the anti-tumor effect of ONC201/TIC10 involves inhibition Akt and ERK phosphorylation resulting in Foxo3a activation and TRAIL-gene transcription. We observed inhibition of Akt phosphorylation upon ONC201/TIC10 treatment of ALL cells. Thus, ONC201/TIC10 holds promise as a novel agent for the treatment of ALL based on its robust activity in preclinical models of the disease. Our ongoing studies are evaluating the impact of this novel therapy on ALL cells with different translocations, and are introducing combination therapy with ONC201/TIC10 for ALL. [Display omitted] DisclosuresAllen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Patents & Royalties.

Transcription Regulation Targeting in Peripheral T Cell Lymphomas Induces Apoptosis and Sensitization to BCL2 Inhibitors

Peripheral T-cell lymphomas (PTCL) are clinically aggressive diseases with poor response to available (largely B-cell lymphoma–tailored) chemotherapy regimens and dismal survival.To identify active drugs for PTCL patients, we performed a cell-based progressive screen from a library of 105 anti-neoplastic drugs in clinical use. Primary screening was done in the PTCL-NOS cell line (OCI-Ly12), using three drug concentrations. We identified 3 active drug groups within the clinical-range limit: HDAC inhibitors (HDI) (romidepsin), proteasome inhibitors (bortezomib, carfilzomib) and transcription inhibitors (dactinomycin). Secondary screening was conducted for the active drug groups with 6 drug concentrations, in an extended panel of 9 TCL cell lines. We also expanded the drugs in each group by adding vorinostat, panobinostat and valproic acid for HDI and SNS032 (CDK9>2>>7 inhibitor) and THZ1 (CDK7>12 inhibitor) for transcription inhibitors. We demonstrated that the most active drugs (within serum achievable concentrations) were bortezomib, carfilzomib, romidepsin, dactinomycin and THZ1. Since proteasome inhibitors and romidepsin are being currently tested in PTCL clinical trials, we focused on the transcriptional inhibitors. Actinomycin inhibits the transcription initiation complex and prevents RNA elongation by RNA-POL2; given this broad mechanism of action, it is associated with serious side effects that limit its clinical use either alone or in combination. On the other hand, a recently discovered CDK7>12 inhibitor, THZ1, showed minimal side effects in vivo (Kwiatkowski N, et al. Nature 2014 Jun22), and high potency in our secondary screening yielding IC50s in TCL of ~200nM. We therefore used THZ1 to investigate the functional relevance of CDK7/12 targeting in PTCL. CDK7 is a critical component of both CDK-activating kinase (CAK) and transcription factor II human (TFIIH) complexes that phosphorylates cell cycle CDKs and C-terminal domain (CTD) of RNA-POL2, respectively. CDK7 inhibition decreases the phosphorylation of RNA-POL2 at Ser5 and Ser2, leading to transcriptional inhibition of susceptible loci. CDK12 in complex with CCNK displays a CTD kinase activity that is required for RNA splicing. To better understand the sensitivity of TCL to THZ1, we first determined the expression of all CAK components (CDK7, CCNH, and MAT1A) and CDK12/CCNK in 9 TCL cell lines that were found overexpressed compared to normal T cells from tonsils. Treatment of OCI-Ly12 and OCI-Ly13.2 (ALCL-ALK negative) cell lines with THZ1 for up to 48 h induced dose- and time-dependent decrease of phospho-POL2-ser5 and phospho-POL2-ser2, followed by PARP cleavage, caspase 7/3 activation and apoptosis. Genes with super-enhancers were found to be more susceptible to THZ1, and we also found these enhancers in genes associated with PTCL prognosis such as MCL1, JAK1 and MYC. Accordingly, THZ1 decreased mRNA and protein levels of MCL1, JAK1 and MYC as early as 3 hours after treatment. This was followed by decreasing levels of BCL2, BCL-XL, JUND and NFkB, and increased expression of pro-apoptotic proteins such as BAX at later time points. The decrease in JAK1 abundance led up to 70% reduction of phospho-STAT3 activity (as determined by immunoblotting and EMSA-like assays). Moreover, induction of STAT3 phosphorylation by IL-7/IL-15 treatment partially rescued the effect of THZ1, suggesting that JAK1 is a relevant target of CDK7 in PTCLs. The THZ1-dependent decrease in anti-apoptotic BCL2-family members prompted us to determine whether CDK7/12 inhibition can re-sensitize TCL to these drugs. We found either synergistic or re-sensitization effects on combination of THZ1 with BH3-family inhibitors, ABT-737 and obatoclax, in both OCI-LY12 and OCI-Ly13.2 cells.In sum, we identified a mechanism by which CDK7/12 inhibition, with the irreversible clinical candidate compound THZ1, simultaneously inhibits prominent PTCL survival pathways (JAK/STAT3, MYC and BCL2) causing apoptosis and re-sensitization to BCL2-family inhibitors. DisclosuresOff Label Use: THZ1 (CDK7 and CDK12 inhibitor) for inducing apoptosis and sensitization to BCL2 inhibitors in peripheral T cell lymphomas.. Kwiatkowski:Syros Pharmaceuticals: One of the inventors Patents & Royalties. Zhang:Syros Pharmaceuticals: One of the inventors Patents & Royalties. Gray:Syros Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

MiR-363 sensitizes cisplatin-induced apoptosis targeting in Mcl-1 in breast cancer.

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic Bcl-2 family member that is often overexpressed in breast tumors, and has been reported to have an important role in regulating drug resistance in various types of cancer including breast cancer. However, the mechanisms underlying the aberrant expression of Mcl-1 are still unclear. In this study, we used bioinformatics, cellular, and molecular methods to predict and prove that miR-363 directly targeted Mcl-1 3'-UTR (3'-untranslated regions) and caused downregulation of Mcl-1 in breast cancer. Resistance to chemotherapy is a major barrier for the effective treatment for advanced breast cancer, but our study indicated that miR-363 reversed the resistance of the breast cancer cell line MDA-MB-231 to the chemotherapeutic agent cisplatin (CDDP). In addition, transfection of breast cancer cells with Mcl-1 expression plasmid abolished the sensitization effect of miR-363 to cisplatin-inducing cytotoxicity. In summary, our study showed that miR-363 was a negative regulator of Mcl-1 expression, and the combination of miR-363 and cisplatin may be a novel approach in the treatment for breast cancer.

Induction of endoplasmic reticulum stress as a strategy for melanoma therapy: is there a future?

Melanoma cells employ several survival strategies, including induction of the unfolded protein response, which mediates resistance to endoplasmic reticulum (ER) stress-induced apoptosis. Activation of oncogenes specifically suppresses ER stress-induced apoptosis, while upregulation of ER chaperone proteins and antiapoptotic BCL-2 family members increases the protein folding capacity of the cell and the threshold for the induction of ER stress-induced apoptosis, respectively. Modulation of unfolded protein response signaling, inhibition of the protein folding machinery and/or active induction of ER stress may thus represent potential strategies for the therapeutic management of melanoma. To this aim, the present article focuses on the current understanding of how melanoma cells avoid or overcome ER stress-induced apoptosis, as well as therapeutic strategies through which to harness ER stress for therapeutic benefit.

Low fucose containing bacterial polysaccharide facilitate mitochondria-dependent ROS-induced apoptosis of human lung epithelial carcinoma via controlled regulation of MAPKs-mediated Nrf2/Keap1 homeostasis signaling.

Reactive oxygen species (ROS), the key mediators of cellular oxidative stress and redox dysregulation involved in cancer initiation and progression, have recently emerged as promising targets for anticancer drug discovery. Continuous free radical assault upsets homeostasis in cellular redox system and regulates the associated signaling pathways to mediate stress-induced cell death. This study investigates the dose-specific pro-oxidative behavior of a bacterial fucose polysaccharide, which attenuated proliferation of different cancer cells. In the fermentation process, Bacillus megaterium RB-05 [GenBank Accession Number HM371417] was found to biosynthesize a polysaccharide with low-fucose content (4.9%), which conferred the maximum anti-proliferative activity (750 µg/mL) against human lung cancer epithelial cells (A549) during preliminary screening. Structural elucidation and morphological characterization of the duly purified polysaccharide was done using HPLC, GC-MS, (1)H/(13)C NMR, and microscopy. The polysaccharide exhibited concentration- and time-dependent anti-proliferative effects against A549 cells by inducing intracellular ROS level and regulating the mitochondrial membrane-permeability following the apoptotic pathway. This process encompasses activation of caspase-8/9/3/7, increase in the ratio of Bax/Bcl2 ratio, translocation of Bcl2-associated X protein (Bax) and cytochrome c, decrease in expression of anti-apoptotic members of Bcl2 family, and phosphorylation of mitogen activated protein kinases (MAPKs). Apoptosis was attenuated upon pretreatment with specific caspase-inhibitors. Simultaneously, during apoptosis, the ROS-mediated stress as well as activated MAPKs triggered nuclear translocation of transcription factors like nuclear factor (erythroid-derived)-like 2 (Nrf2) and promoted further transcription of downstream cytoprotective genes, which somehow perturbed the chemotherapeutic efficacy of the polysaccharide, although using CuPP, a chemical inhibitor of HO-1, apoptosis increased significantly (P < 0.05).

Mad2 and BubR1 modulates tumourigenesis and paclitaxel response in MKN45 gastric cancer cells

Aneuploidy and chromosomal instability (CIN) are common features of gastric cancer (GC), but their contribution to carcinogenesis and antitumour therapy response is still poorly understood. Failures in the mitotic checkpoint induced by changes in expression levels of the spindle assembly checkpoint (SAC) proteins cause the missegregation of chromosomes in mitosis as well as aneuploidy. To evaluate the possible contribution of SAC to GC, we analyzed the expression levels of proteins of the mitotic checkpoint complex in a cohort of GC cell lines. We found that the central SAC proteins, Mad2 and BubR1, were the more prominently expressed members in disseminated GC cell lines. Silencing of Mad2 and BubR1 in MKN45 and ST2957 cells decreased their cell proliferation, migration and invasion abilities, indicating that Mad2 and BubR1 could contribute to cellular transformation and tumor progression in GC. We next evaluated whether silencing of SAC proteins could affect the response to microtubule poisons. We discovered that paclitaxel treatment increased cell survival in MKN45 cells interfered for Mad2 or BubR1 expression. However, apoptosis (assessed by caspase-3 activation, PARP proteolysis and levels of antiapoptotic Bcl 2-family members), the DNA damage response (assessed by H2Ax phosphorylation) and exit from mitosis (assessed by Cyclin B degradation and Cdk1 regulation) were activated equally between cells, independently of Mad2 or BubR1-protein levels. In contrast, we observed that the silencing of Mad2 or BubR1 in MKN45 cells showed the induction of a senescence-like phenotype accompanied by cell enlargement, increased senescence-associated β-galactosidase activity and increased IL-6 and IL-8 expression. In addition, the senescent phenotype is highly increased after treatment with PTX, indicating that senescence could prevent tumorigenesis in GC. In conclusion, the results presented here suggest that Mad2 and BubR1 could be used as prognostic markers of tumor progression and new pharmacological targets in the treatment for GC.

HTLV-1 tax stabilizes MCL-1 via TRAF6-dependent K63-linked polyubiquitination to promote cell survival and transformation.

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein hijacks the host ubiquitin machinery to activate IκB kinases (IKKs) and NF-κB and promote cell survival; however, the key ubiquitinated factors downstream of Tax involved in cell transformation are unknown. Using mass spectrometry, we undertook an unbiased proteome-wide quantitative survey of cellular proteins modified by ubiquitin in the presence of Tax or a Tax mutant impaired in IKK activation. Tax induced the ubiquitination of 22 cellular proteins, including the anti-apoptotic BCL-2 family member MCL-1, in an IKK-dependent manner. Tax was found to promote the nondegradative lysine 63 (K63)-linked polyubiquitination of MCL-1 that was dependent on the E3 ubiquitin ligase TRAF6 and the IKK complex. Tax interacted with and activated TRAF6, and triggered its mitochondrial localization, where it conjugated four carboxyl-terminal lysine residues of MCL-1 with K63-linked polyubiquitin chains, which stabilized and protected MCL-1 from genotoxic stress-induced degradation. TRAF6 and MCL-1 played essential roles in the survival of HTLV-1 transformed cells and the immortalization of primary T cells by HTLV-1. Therefore, K63-linked polyubiquitination represents a novel regulatory mechanism controlling MCL-1 stability that has been usurped by a viral oncogene to precipitate cell survival and transformation.

MCL1 and BCL-xL levels in solid tumors are predictive of dinaciclib-induced apoptosis.

Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclib's antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.

Catechins induced acute promyelocytic leukemia cell apoptosis and triggered PML-RARα oncoprotein degradation.

BackgroundIt has recently been reported that the extracts of green tea polyphenol have cancer preventive effects. In this study, we investigated the effect of the natural composition from green tea leaves Catechins on acute promyelocytic leukemia (APL).MethodsIn vitro, APL cell lines NB4, retinoic acid-resistant NB4-R1 and NB4-R2 were treated with different concentrations of Catechins. Cell viability and cell apoptosis were analyzed using MTT assay and flow cytometric assay, respectively. Expression of proteins related to apoptosis and PML-RARα oncoprotein were assessed by Western blot. In vivo anti-tumor activity of Catechins was examined in nude mice xenografted with NB4 cells and in situ cell apoptosis was detected by terminal deoxytransferase-catalyzed DNA nick-end labeling assay.ResultsCatechins at micromolar concentration levels significantly inhibited APL cell proliferation and induced cell apoptosis, in association with mitochondria damage, ROS production and caspase activation. The anti-apoptotic Bcl-2 family member Bcl-xL was down regulated, with pro-apoptotic member Bax remaining unchanged. Moreover, Catechins induced the degradation of PML-RARα oncoprotein. Catechins-mediated apoptotic effect was also observed in primary APL cells without affecting normal hematopoietic progenitor cells. In the murine xenograft model, Catechins remarkably inhibited tumor growth and induced in situ leukemic cell apoptosis.ConclusionsCatechins might be a potential candidate for APL treatment by activating intrinsic apoptotic pathway and targeting PML-RARα oncoprotein.

The effects of dexamethasone on the proliferation and apoptosis of human ovarian cancer cells induced by paclitaxel.

BackgroundDexamethasone (DEX) has been routinely used as a pre-treatment in the clinical application of paclitaxel (PTX) to treat ovarian cancer. However, PTX-induced apoptosis might be inhibited by DEX. This study was undertaken to investigate the effects of DEX on the apoptosis induced by PTX.MethodsBoth of SKOV-3 and HO-8910 human ovarian cancer cells were divided into four groups: (1) untreated (Con); (2) treated with DEX (0.1 μM) alone; (3) treated with PTX (50 nM); and (4) pre-treated with DEX (0.1 μM), and 24 h later, treated with PTX (DEX + PTX). Cell proliferation was determined by the 3-(4,5)-dimethylthiahiazo (−z-y1)-3,5-di- phenytetrazoliumromide (MTT) dye uptake method, while cell apoptosis was analyzed by propidium iodide (PI) staining and flow cytometry. Then, reverse transcription polymerase chain reactions (RT-PCRs) were applied to semi-quantitative analysis, followed by western blot analysis. Statistical analysis was performed, with Fisher’s least significant difference test.ResultsOur results demonstrated that DEX can differentially inhibit SKOV-3 and HO-8910 cell proliferation induced by PTX and decrease the apoptosis rates in cancer cells. Pre-treatment with DEX could up-regulate the expressions of members of anti-apoptotic Bcl-2 family (Bcl-2 and Bcl-XL) and members of IAP family (survivin). The expression of cleaved caspase-3 was down-regulated by DEX, shown by semi-quantitative RT-PCRs and western blot analysis.ConclusionsOur data gained invaluable insights of the antagonistic mechanisms of DEX on PTX-induced cancer cell death and may provide new methods of using DEX as antineoplastic drugs or agents in the clinical treatment for ovarian cancer patients.

Abstract 2961: The level of mitochondrial apoptotic priming determines cell fate upon p53 restoration

Abstract Using genetically engineered mouse models of cancer, we and others have recently shown that a variety of established tumors require sustained inactivation of the p53 pathway. Reactivation of p53 in established tumors typically results in one of two cell fate decisions: cell cycle arrest or cell death via apoptosis. Since p53 is a transcription factor, one hypothesis that may explain these tumor-specific outcomes is that p53 transcriptionally activates a particular set of genes that is specific to each tumor type, in addition to genes involved in a core p53 pathway. Another possibility is that the level of mitochondrial apoptotic priming in a cell might dictate whether p53 activation promotes cell death or cell cycle arrest. To test this, we derived murine p53-restorable cell lines from three different tumor types: lung adenocarcinoma, sarcoma and lymphoma. Upon restoration of the endogenous Trp53 gene, these cell lines undergo cell cycle arrest (lung and sarcoma) or apoptosis (lymphoma). We hypothesized that this tumor-specific response to p53 restoration could be explained by the level of mitochondrial priming on each specific tumor type, where lymphoma cell lines would be highly primed relative to the lung and sarcoma cell lines. Indeed mitochondrial BH3 profiling demonstrated that lymphoma cell lines were highly primed and this correlated with their apoptotic response to p53 restoration. On the other hand, lung and sarcoma cell lines were poorly primed and this was consistent with their cell cycle arrest phenotype upon p53 restoration. Modulating the expression levels of Bcl2 family members to modulate mitochondrial apoptotic priming was sufficient to change the fate of these cells. Lymphoma cell lines were forced to undergo cell cycle arrest upon p53 restoration by overexpression of the anti-apoptotic Bcl2 family members Bcl-2 or Bcl-XL. In contrast, lung and sarcoma cell lines were forced to undergo cell death upon p53 restoration when priming was increased by overexpression of the pro-apoptotic Bcl2 family members Bim or Bad. When transplanted subcutaneously, sarcoma cell lines expressing Bad were specifically sensitized to undergo cell death upon p53 restoration whereas cell lines expressing empty vector underwent cell cycle arrest. We are currently carrying out in vivo experiments with lymphoma cell lines and lung cell lines, as well as with autochthonous tumor models and human cell lines to further test our main hypothesis. These findings may offer key insights into the potential use of drugs that restore p53 function. Citation Format: Francisco J. Sánchez-Rivera, Jeremy Ryan, Yadira M. Soto-Feliciano, David M. Feldser, Michael T. Hemann, Anthony Letai, Tyler Jacks. The level of mitochondrial apoptotic priming determines cell fate upon p53 restoration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2961. doi:10.1158/1538-7445.AM2014-2961

Abstract 4600: P-TEFb is a therapeutic target in human bortezomib-resistant multiple myeloma cells

Abstract Despite proteasome inhibitor (e.g., bortezomib/btz and carfilzomib/cfz) success in multiple myeloma (MM), drug resistance remains a key problem. MM cells commonly over-express Mcl-1, a protein implicated in survival and drug-resistance. Mcl-1 exhibits rapid protein turnover (half-life &amp;lt; 1h), suggesting that efficient transcriptional machinery for de novo synthesis may be required for its constitutive expression in MM. Eukaryotic protein-coding gene transcription is controlled by RNA polymerase II (RNAPII), which is regulated by negative (N-TEF) and positive elongation factors (P-TEF). P-TEFb, a holoenzyme Cdk9/cyclin T complex, activates RNAPII by phosphorylating the S2 site within the carboxy-terminal domain (CTD). Here, the role of P-TEFb in Mcl-1 maintenance was investigated in MM cells. Anti-apoptotic Bcl-2 family members profiling revealed high Mcl-1 protein levels in virtually all MM lines tested and up-regulation in drug-resistant lines (e.g., btz-resistant PS-R cells). Similar patterns occurred in human MM xenograft mouse models and primary CD138+ MM cells. Ectopic Mcl-1 over-expression strikingly diminished btz lethality, arguing that Mcl-1 is a critical determinant of btz-resistance. All tested MM cell lines, primary MM specimens, and murine xenograft samples displayed constitutive P-TEFb activation i.e., high CTD (S2) phosphorylation, which correlated with a) up-regulation of P-TEFb subunits i.e., Cdk9 (42 and 55kD isoforms) and its partner cyclin T1 (but not T2a/b or K); b) marked 42kD T186 phosphorylation, while drug-resistant cells also displayed sharply increased 55kD T186 phosphorylation; and c) constitutive P-TEFb complex assembly and RNAPII interactions, but little inhibitory association with HEXIM which binds to P-TEFb via 7SK snRNP. The function of P-TEFb in Mcl-1 regulation was then validated by genetic and pharmacological strategies. First, Cdk9 or cyclin T1 shRNA knock-down dramatically inhibited CTD S2 phosphorylation and down-regulated Mcl-1. Second, pan-Cdk inhibitors e.g., dinaciclib rapidly inhibited P-TEFb and down-regulated Mcl-1, leading to apoptosis prior to cell cycle perturbations. These events primarily reflected P-TEFb complex disassembly, but not levels of P-TEFb subunits or T186 phosphorylation. Third, like Mcl-1 knock-down, Cdk9 shRNA or a specific Cdk9 inhibitor significantly restored susceptibility of btz-resistant cells to btz or cfz, and also sensitized them to the BH3-mimetic ABT-737 which does not target Mcl-1. Finally, pan-Cdk inhibitors reduced tumor burden and eliminated human CD138+ cells in the bone marrow of murine models bearing drug-naïve or btz-resistant MM cells. Collectively, these findings implicate constitutive activation of P-TEFb in maintenance of high Mcl-1 levels in MM, and argue that P-TEFb may serve as a rational target to overcome btz-resistance. This study was supported by the MD Anderson SPORE in Myeloma. Citation Format: Yun Dai, Shuang Chen, Liang Zhou, Yu Zhang, Yun Leng, Xin-Yan Pei, Hui Lin, Robert Z. Orlowski, Steven Grant. P-TEFb is a therapeutic target in human bortezomib-resistant multiple myeloma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4600. doi:10.1158/1538-7445.AM2014-4600